Natural Variation for Lifespan and Stress Response in the remanei

Rose M. Reynolds¤, Patrick C. Phillips* Institute of Ecology and Evolution, University of Oregon, Eugene, Oregon, United States of America

Abstract Genetic approaches (e.g. mutation, RNA interference) in model organisms, particularly the nematode , have yielded a wealth of information on cellular processes that can influence lifespan. Although longevity mutants discovered in the lab are instructive of cellular physiology, lab studies might miss important genes that influence health and longevity in the wild. C. elegans has relatively low natural genetic variation and high levels of linkage disequilibrium, and thus is not optimal for studying natural variation in longevity. In contrast, its close relative C. remanei possesses very high levels of molecular genetic variation and low levels of linkage disequilibrium. To determine whether C. remanei may be a good model system for the study of natural genetic variation in aging, we evaluated levels of quantitative genetic variation for longevity and resistance to oxidative, heat and UV stress. Heritability (and the coefficient of additive genetic variation) was high for oxidative and heat stress resistance, low (but significant) for longevity, and essentially zero for UV stress response. Our results suggest that C. remanei may be a powerful system for studying natural genetic variation for longevity and oxidative and heat stress response, as well as an informative model for the study of functional relationships between longevity and stress response.

Citation: Reynolds RM, Phillips PC (2013) Natural Variation for Lifespan and Stress Response in the Nematode Caenorhabditis remanei. PLoS ONE 8(4): e58212. doi:10.1371/journal.pone.0058212 Editor: Matt Kaeberlein, University of Washington, United States of America Received September 3, 2012; Accepted February 1, 2013; Published April 26, 2013 Copyright: ß 2013 Reynolds, Phillips. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work received funding support from the National Institutes of Health (R03-AG029377 and P01-AG022500 to PCP; Ruth L. Kirschstein National Research Service Award (NRSA) Postdoctoral Fellowship AG032900 to RMR), and the Ellison Medical Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] ¤ Current address: Department of Biology, William Jewell College, Liberty, Missouri, United States of America

Introduction selection experiments. One notable advantage of C. elegans over D. melanogaster is that it can be frozen, allowing parental and Current understanding of the genetic basis of the aging process descendant generations to be tested at the same time and allowing has been formed mainly by mutagenesis and RNA interference large numbers of lines to be maintained at minimal effort. Despite studies in the model systems Saccharomyces cerevisiae (yeast), these advantages, the C. elegans model is not optimal for studies of Caenorhabditis elegans (soil-dwelling roundworm), and Drosophila natural genetic variation. Recently several studies have shown that melanogaster (common fruit fly) [1]. Most loci identified as affecting C. elegans populations possess low levels of molecular genetic lifespan in those species are members of evolutionarily conserved variation on a worldwide scale [9–11]. Worse, nearly all of the metabolic pathways such as the insulin/insulin-like signaling [2], existing variation is locked within -wide linkage disequi- target-of-rapamycin [3], Jun kinase [4] and sirtuin [5] pathways. librium, yielding a small number of effective haplotypes [11–13]. ‘‘Age mutants’’ can be defined as those displaying extreme Few studies have estimated heritability of lifespan in C. elegans, but longevity, falling well above the maximum lifespan observed under published estimates are not significantly different from zero [14]. normal laboratory conditions. Although natural populations do These issues suggest that C. elegans will serve as a poor model for not display the extreme longevity achieved by Age mutants, studying variation in aging within natural populations [15]. quantitative genetic research has revealed that there is significant In contrast, a close relative of C. elegans, C. remanei, may be an and heritable variation in lifespan within genetically heteroge- ideal system in which to study natural genetic variation. Unlike the neous populations, e.g. h2 = 0.03 [6] to h2 = 0.73 [7]. This implies low genetic variation found within the selfing C. elegans, that studies of the underlying genetics of aging in natural populations of C. remanei harbor significant amounts of molecular populations may suggest promising molecular targets for inter- genetic variation [16,17], and linkage disequilibrium breaks down vention in age-related disease and dysfunction. on the order of 100 base pairs [18]. Unlike the androdioecious C. C. elegans has been a workhorse in the genetics of aging: elegans, C. remanei is composed of males and females, making it a mutagenesis and gene expression studies in C. elegans have better model for human aging. It is a non-parasitic, soil-dwelling facilitated a detailed understanding of many longevity genes and their roles within key metabolic pathways [8]. Its genome has been roundworm that is easily collected in the wild. Significantly, it can sequenced and assembled, it has very short generation times (3–4 be frozen within just a few generations of collection, thereby days), and it can be cultured at extremely large population sizes, maintaining a majority of the natural genetic variation present in facilitating the construction of genetic lines and the conduction of the population when in its natural habitat. Like C. elegans, its

PLOS ONE | www.plosone.org 1 April 2013 | Volume 8 | Issue 4 | e58212 Natural Variation for Lifespan in C. remanei genome has been sequenced, it has short generation times (,4 three to five randomly selected L4 females (dams) from the same days), can be cultured at large population sizes, can be genetically stock. Each sire was given 24 hours to sexually mature and mate transformed, and can be frozen, giving us the ability to compare with dams. We then placed each dam on her own small plate and evolved or manipulated genetic lines to the original ancestral allowed her to lay eggs for 24 hours, after which she was population. discarded. Because each dam mated with only one sire, all Given the large amount of molecular genetic variation offspring from a single dam were full sibs; offspring from different previously found in C. remanei for a variety of molecular pathways dams mated to the same male were half-sibs. From each dam, we [17], we predicted that C. remanei might possess the genetic collected eight to 16 full-sib L4 male and female offspring and variation for longevity and stress resistance necessary to make it a placed them on seeded NGM plates in same-sex groups overnight. good model system for the study of natural variation in aging. 20 to 24 hours later those offspring had become young adults and Specifically, we looked for evidence of significant natural genetic were then used in either an oxidative or UV shock assay as variation for longevity and acute heat, oxidative and UV stress described below. For each sire, the offspring from three separate resistance in a genetically heterogeneous population of C. remanei. dams were tested (3 half-sib families). We found high heritability for oxidative and heat stress resistance, Parent-Offspring Design. We randomly collected 120 L4 low but significant heritability for lifespan, and no heritability for females and 360 male L4s (grandparental generation) from the UV stress resistance. These results suggest that C. remanei may be a genetically heterogeneous PX323 stock. Each female was placed good model in which to look for natural genetic variants increasing with three males. These mating groups were given 24 hours to health and lifespan. In particular, it appears promising for sexually mature and mate, after which all adults were discarded. investigations into the complex relationship between stress Female offspring (dams) were collected as L4s within a single six- response and longevity. hour window. Each dam was given three L4 sires (randomly selected from the PX323 stock) with which to mate. 24 hours later Materials and Methods sires were discarded and dams were moved to new plates daily until reproduction had ceased, then once per week until death by Strains & Handling natural causes. Three daughters were collected per female. Each Wild C. remanei were isolated from terrestrial isopods (woodlice) L4 daughter was given three L4 males (randomly selected from the from a single population near Dayton, Ohio and generously PX323 stock) with which to mate. 24 hours later, males were supplied to us by Scott Baird (Wright State University) with the discarded and daughters were moved to new plates daily until permission of the State of Ohio Department of Natural Resources reproduction had ceased, and then once per week until death by (see [19] for details). Isofemale strains were created from mating natural causes. Under this design, daughter lifespans (daughters pairs and inbred for 10 generations to create distinct genetic could be full or half sibs) were regressed against dam lifespan. strains. Those strains were inbred via full-sib mating, and then individually frozen for long-term storage. We obtained nineteen of Phenotyping & Analysis those strains (PB strains 234, 237, 241, 244, 245, 247, 254, 255, Longevity. We estimated genetic variation for longevity using 256, 257, 258, 259, 261, 263, 266, 269, 271, 272, 285), added both strain-based and parent-offspring regression methods. For them in equal proportions to the same Petri plate with media, and both methods we maintained worms individually on small NGM- allowed them to randomly mate for four generations in order to Lite plates that were seeded with OP50. All worms were generate a genetically heterogeneous C. remanei population. This transferred to freshly seeded plates at weekly intervals. Longevity genetically heterogeneous population (PX323) was frozen at was determined by checking each individual daily at the same time 280uC for future use (as were all strains used in this study). To of day. Worms were declared dead if they did not respond to being measure heritable genetic variation for longevity and traits that prodded with a platinum wire. may be correlated with longevity, we employed half-sib, strain- We performed statistical analyses using JMP 8.0 and SAS 9.2. based, and parent-offspring regression approaches to measure [20]. We determined the significance of strain-specific differences genetic variation and estimate trait- and sex-specific heritability for in mortality using the Cox proportional hazards model with main various traits. All strains were thawed and given two generations to effects of strain and replicate within strain (not all strains were in recover from freeze before collecting experimental individuals. all replicates). When estimating genetic variance for mean lifespan, Strains were maintained on NGM-Lite agar plates seeded with individuals who had left the agar plates or been accidentally killed Escherichia coli (OP50) as a food source. All populations were (right censored) were removed from the data set and genetic housed in an incubator at 20uC after thaw and throughout the variance was determined using an analysis of variance including experiment. the random effects of strain and replicate within strain. Heritability 2~ 2 2 z 2 2 Inbred Strain Design. To obtain stage-synchronized indi- was calculated ash (sAS=2) (sAS=2 sWS), where sAS is the 2 viduals for inbred strain-based measures of genetic variation, we among-strain variance component and sWS is the within-strain randomly collected one female and one male fourth stage larvae variance component [21]. Average N per strain: females = 35.9, (‘‘L4,’’ virgin) from each of ten isofemale strains (PB lines 234, 241, males = 28.9. To confirm our strain-based estimate of heritability 244, 245, 261, 266, 269, 271, 272). These mating pairs were given we performed a parent-offspring regression on the data collected 24 hours to sexually mature and mate. Females were then moved as described above. We performed a general linear regression of to individual plates and allowed to lay eggs for 6–24 hours, after daughter lifespan against parent (dam) lifespan. All lifespan data which all adults were discarded. Offspring were collected as L4s were log10 transformed to normalize residuals, and outliers were within a single six-hour window, and placed on seeded plates in removed, N = 73 families. Standard errors on the estimates and groups of 10–15 overnight. 20 to 24 hours later the offspring had significance tests were conducted via bootstrap analysis [22]. become young adults and were then used in either a heat shock or Heat Stress Resistance. We performed extensive prelimi- a longevity assay as described below. nary testing to determine a temperature and length of exposure Half-sib Design. For each assay, ten males (sires) were such that the genetically heterogeneous strain (PX323, a proxy for collected as L4s from the genetically heterogeneous PX323 stock. mean isofemale line response) experienced approximately 50% We housed each sire on his own small (35610 mm) plate with survival. We observed that thermal maximum for males is at least

PLOS ONE | www.plosone.org 2 April 2013 | Volume 8 | Issue 4 | e58212 Natural Variation for Lifespan in C. remanei one full degree Celsius lower than that for females. Within sex, a data and evaluating the extent of the overlap of the resulting change in temperature of half a degree made the difference distributions was via a Mann-Whitney U test. between 80% and 0% survival (unpublished data). Therefore, we chose to test only females at this time. Results L4 females were collected from ten of the nineteen isofemale strains (PB strains 234, 237, 241, 244, 245, 255, 258, 269, 271 and Mean lifespan significantly differed among lines (analysis of 272; strains were selected at random) as described above, and variance; Females F10, 280 = 4.60, P,0.0001; Males F10, 280 = 4.60, housed in same-sex groups of 10–12 on small, seeded NGM plates P,0.0001; Figure 1a,b; Table S1), as did the age-specific risk of 2 for 24–30 hours. We then placed them in strain-specific groups of death (Cox proportional hazard; Females x10 = 83.04, P,0.0001; 2 60 one-day-old virgin adults onto large, seeded NGM plates and Males x7 = 25.76, P = 0.0006; Figure 1C & D). For females, CVA 2 exposed them to 35.5uC for 16 hours. We then moved the worms and h – measures of the additive genetic component of the to 20uC and allowed them to recover for three hours before differences in lifespan among lines – were small, but significantly scoring plates for live and dead worms. Genetic variance for heat greater than zero (Table 1), and within the range of published stress resistance ability was determined using an analysis of estimates for other organisms [6]. In contrast, for males neither 2 variance with random effects of strain and replicate. Heritability CVA nor the line-based or parent-offspring estimates of h for was calculated as for lifespan. Data were square root transformed lifespan were significantly from zero (Table 1; Figure S1). to meet distribution assumptions for the analysis of variance. The ability to withstand heat stress differed significantly among Average replication (N) per strain = 8. Three of ten strains had lines (Females F6,6 = 12.06, P = 0.004; Figure 2; Table S2) and 2 fewer than six replicates and were therefore excluded from the showed significant heritability in our population. Indeed, h for data set during analysis. heat stress resistance was more than five times greater than that for Oxidative Stress Resistance. Preliminary tests suggested lifespan (Mann-Whitney U, P,0.0001, Table 1). This pattern was that moderate differences in resistance among PX323 families further reflected when comparing CVA between the two traits: CVA would be detectable if oxidant-induced mortality caused 100% for heat stress was nearly twice that for longevity. death between four and eight hours. As for heat stress resistance, Likewise, the ability to withstand oxidative stress was signifi- males were less resistant to hydrogen peroxide than were females, cantly different among the offspring of different sires (Cox 2 but the difference in resistance between sexes was not extreme, proportional hazard; Females x7 = 85.87, P,0.0001; Males 2 therefore we tested both sexes using a single concentration and x9 = 36.54, P,0.0001; Figure 3; Table S3). Heritability for duration of exposure to hydrogen peroxide. oxidative shock resistance was the highest of the traits measured We placed one-day-old virgin adults into either 40 mLof in our study; in females it was two times greater than that for heat 3.5 mM H2O2 (experimental individuals) or 40 mL of S-basal stress resistance and more than 11 times greater than that for (controls). Worms were checked every 30 minutes until all lifespan (Mann-Whitney U P,0.0001, Table 1). CVA estimates for experimental individuals failed to respond to prodding with a oxidative and heat stress resistances were comparable. platinum wire for three continuous samplings (scored dead for 90 The ability to withstand UV stress (days survived after exposure 2 minutes). Estimates of genetic variance for oxidative stress to 2000 J/m ) was not significantly different among offspring of resistance ability was determined using analysis of variance model different sires (Females F9 = 1.33, P = 0.22; Males = F9 = 1.71, with random effects of sire, dam within sire, and replicate. P = 0.09). Despite some differences in age-specific mortality (Cox 2~ 2 2 2 proportional hazard; Females x2 = 16.41, P = 0.06; Males Heritability was calculated as h sA sP, where sA is additive 9 2 x2 = 23.89, P = 0.04; Figure 4), we found no additive genetic genetic variance and sP is total phenotypic variance [23]. 9 UV Stress Resistance. Preliminary tests suggested that variance for resistance to UV exposure, and heritability for the moderate differences in resistance among PX323 families would trait was likewise zero (Table 1; Table S4). Mean survival after UV be detectable if UV exposure-induced mortality caused 100% exposure was significantly different between males and females death within one to two weeks. As for oxidative stress resistance, (F1,460 = 99.84, P,0.0001) as was age-specific mortality (Cox 2 males were less resistant to UV stress than were females, but the proportional hazard; x1 = 51.50, P,0.0001, Figure 4) with females difference in resistance between sexes was not extreme, therefore living surviving longer on average (Females 6.7260.18 days; Males we tested both sexes using a single duration of exposure to UV 4.9860.12 days). Males were less stress resistant to UV stress than irradiation. were females. We placed one-day-old adults with their same-sex full sibs onto large unseeded plates and exposed to 2000 J/m2 UV irradiation Discussion (FisherBiotech Microprocessor-Controlled UV Crosslinker FB- UVXL-1000 using 254 nm UV tubes). Worms were immediately Contrasting patterns of natural genetic variation for picked to large seeded plates and checked daily until all worms longevity and stress resistance failed to respond to prodding with a platinum wire. Estimates of We have shown that C. remanei has heritable genetic variation for genetic variance for UV stress resistance ability was determined lifespan (significant for females), and high heritability for two using analysis of variance with random effect of sire, dam within stressors hypothesized to be linked to longevity: heat and oxidative sire, and replicate. Heritability was calculated as for oxidative stress resistance. Heat, oxidative and UV stress resistance are all stress response. traits previously correlated with longevity in Age mutants [26]. Of Calculation of CVA and errors. For all traits, the coefficient the three, oxidative stress resistance has received the most attention in the aging literature, being a hypothesized mechanism of additiveqffiffiffiffiffiffi genetic. variance (CVA) was calculated as for senescence. The oxidative stress hypothesis proposes that aging CV ~ 2 2 A 100 sA m, where sA is additive genetic variance and m occurs because of the gradual accumulation of molecular and is the mean of the phenotypic response [24]. Errors on cellular damage caused by chemically reactive molecules, reactive quantitative genetic parameters were calculated as described in oxygen species in particular [27]. A corollary of this hypothesis is [25] where possible, and otherwise by bootstrapping [22]. that oxidative stress resistance ability, through preventative or Comparison of h2 values was accomplished by bootstrapping the reparative means, is a primary determinant of lifespan. Our data

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Figure 1. Differences in lifespan of C. remanei isofemale lines. Top row: age-specific survival (S(t)) of females (A), males (B). Middle row: age- specific hazard (risk of death) of females (C), males (D) Hazard is defined as the instantaneous risk of death. Although hazards have no universal scale, max hazard (100% risk of death within the following day) for these data is one. The reciprocal of the hazard 1/h(t) is expected time to death, given survival to age t. Bottom row: mean adult lifespan of females (E) and males (F); bars are standard errors. Lifespan displayed low but significant heritability and coefficient of additive genetic variation (Table 1). doi:10.1371/journal.pone.0058212.g001

Table 1. Estimates of heritabilities and coefficients of additive genetic variation in C. remanei.

2 Trait Method of estimation Sex h (SE) CVA

Lifespan Inbred Lines Female 0.08(0.04)** 13.07** Inbred Lines Male 0.06(0.05) 12.85 Parent-Offspring Female 0.06(0.29) 9.14 Heat Stress Resistance Inbred Lines Female 0.45(0.18)* 28.59* Male – 1 – 1 Oxidative Stress Resistance Half-Sib Female 0.95(0.55)** 22.05** Male 0.75(0.52)* 17.28* UV Stress Resistance Half-Sib Female 20.05(0.12) 0 2 Male 20.21(0.18) 0 2

*P,0.05, **P,0.01 1Males not assayed. 2Negative variance component estimates. doi:10.1371/journal.pone.0058212.t001

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Figure 2. Mean survival of females from C. remanei isofemale lines after a 16 hour heat stress at 37.56C. Worms were exposed as one-day-old adult virgins. Heritability for heat stress resistance was high (h2 = 0.45, Table 1), and significantly greater than those for longevity or UV stress (Mann-Whitney U, P,0.0001). doi:10.1371/journal.pone.0058212.g002 show dramatically different scales of genetic variation for longevity than for oxidative stress resistance. Although heritability and CVA for longevity were low in this population, they are non-zero, and similar to published estimates for other species [6,28]. Heritability for oxidative stress response in this population of C. remanei was more than 11-fold that for longevity (Females, Table 1). The difference in heritability estimates between longevity and oxidative stress resistance in our study likely reflects the complexity Figure 4. Mortality rates (hazard) after exposure of C. remanei of the genetic relationship between the two traits. The oxidative to 2000 J/m2 UV, plotted by sire. Dashed lines = males, solid stress theory of aging is coming under increasingly critical lines = females. (A) Age-specific survival (S(t)); (B) Age-specific hazard. examination [29]. Most Age mutants are known to confer Max hazard (100% risk of death within the next 30 minutes) for these resistance to oxidative stress, e.g. C. elegans daf-2 [30], and D. data is 1.0. Although there is a large sex effect on oxidative stress resistance, there is no evidence for significant variation among offspring melanogaster mth [31]. Yet, genetic or genomic manipulations of different sires (h2,0.00, Table 1). doi:10.1371/journal.pone.0058212.g004

attempting to reduce oxidative damage, such as over-expression of antioxidants, have either been shown to increase lifespan [32], do nothing [33], or actually reduce lifespan [34]. Reductions of oxidative defenses, such as deletions of stress resistance genes, can result in increased oxidative damage and decreased lifespan [35], but sometimes have no effect [29,36], or have extended lifespan in some cases [37]. Lastly, although some populations of inverte- brates artificially selected for long life are oxidatively resistant [6], not all oxidatively resistant populations are long lived [38]. To some extent, the difference in heritability between these traits can be attributed to the large environmental variance typically observed for longevity, which appears in the denominator of heritability. However, a two-fold difference in genetic variation is still observed when the coefficient of additive genetic variation is use as the measure genetic variation instead of heritability ([24]; Table 1). Overall then, it is becoming clear that, while it may play a significant role in age-related disease, the relationship between oxidative stress resistance and longevity is complex.

Stress-specificity of cellular stress responses Although UV stress resistance is typically correlated with both extended lifespan and oxidative stress resistance in Age mutants [26], we found no genetic variation and thus no heritability for it Figure 3. C. remanei survival during exposure to 3.5 mM H2O2, plotted by sire. (A) Age-specific survival (S(t)) and (B) age-specific in our study population. Our results raise the possibility that, in at hazard for males (dashed lines) and females (solid lines). Max hazard least one rearing environment and genetic background, UV and (100% risk of death within the next 30 minutes) for these data is 0.08. oxidative stress resistance are genetically uncorrelated. This is Control worms (housed in S basal only, not exposed to H2O2) particularly intriguing since there is a large body of literature experienced no death during the assay; mortality not shown. 2 e.g. Heritabilities were high for females and males (h = 0.95 and 0.75 demonstrating that cellular responses to specific stressors ( respectively, Table 1) and were significantly greater than heritability for pathogens, and oxidative, starvation and heat stress) use a limited longevity or UV stress (Mann-Whitney U, P,0.0001). number of common cellular pathways (IIS, TGF-ß, MAPK, JUN- doi:10.1371/journal.pone.0058212.g003 K, HSF-1) and that those cellular responses are controlled through

PLOS ONE | www.plosone.org 5 April 2013 | Volume 8 | Issue 4 | e58212 Natural Variation for Lifespan in C. remanei a likewise limited number of transcription factors (SKN-1, DAF- Supporting Information 16, HSF-1, HIF-1, PHA-4). Indeed, the cellular response to any single stressor appears to be achieved through pathways at least Figure S1 Mean survival time (in days) of C. remanei partially overlapping cellular responses to other stressors [8]. daughters regressed upon lifespan of mothers. All worms were mated to three male C. remanei for 24 hours, and kept under typical laboratory conditions (20C, nematode growth media, lawn Environmental context of expressed variation of E. coli). One possible reason for the difference in heritability estimates (PDF) for longevity and oxidative stress resistance might be that longevity was measured in an artificially benign environment with regard to Table S1 Lifespan data (in days) for virgin C. remanei oxidative challenge. In a more naturalistic spatial and nutritional under typical laboratory conditions. (20C, nematode environment, oxidative stress resistance may indeed determine growth media, lawn of E. coli). lifespan. Empirical studies have measured the heritabilities of traits (PDF) in both stressful and non-stressful environments and found that Table S2 Percent survival of female C. remanei exposed heritabilities of some traits are higher under stressful conditions to 35.56C for 16 hrs. We used 60 one-day-old virgin adults per [29–41]. However, a relatively equal number of findings have trial. been of the opposite effect: that heritability of traits is lower in (PDF) stressful environs than in benign ones [40–42]. In our study, Table S3 Mean survival time (in minutes) of half-sib mortality under oxidative stress was 100% within eight hours of families of C. remanei exposed to 3.5 mM H O until exposure. Because oxidative stress resistance and lifespan are 2 2 death. N is number of offspring tested. inexorably linked in this experiment, it could be said that our stress (PDF) resistance assays were in fact longevity assays under stressful conditions. Regardless of the relationship between levels of stress Table S4 Mean survival time (in days) of half-sib and levels of heritability for a particular trait, we can conclude families of C. remanei exposed to 2000 J/m2 ultraviolet that, for this population of C. remanei, lifespan under relatively irradiation. benign conditions (perhaps most relevant to current human (PDF) lifespan) is not determined by the same suite of genes as is resistance to oxidative stress in the environmental context of this Acknowledgments study. We thank T. Ahearne, H. Archer, M. Byrne, T. Love, S. Mete, S. Scholz, Overall, this study demonstrates that there is significant genetic C. Smith, and A. Stuart Malone for substantial laboratory assistance. We variation for stress-related phenotypes within C. remanei, but that would also like to thank the members of the Phillips laboratory for fruitful the relationship between stress resistance and longevity must be discussions about this work. Strains used in this work were generously complex and environmentally contingent, if it exists at all. This provided by Scott Baird (Wright State University). illustrates that stress and longevity effects observed via mutagenesis may not always be reflected in natural variation; understanding Author Contributions the molecular basis of that variation should be highly informative Conceived and designed the experiments: RMR PCP. Performed the of molecular mechanisms of longevity in its own right. C. remanei experiments: RMR. Analyzed the data: RMR PCP. Wrote the paper: should provide a powerful system for such analyses. RMR PCP.

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